Phosphoprotein preparations for bioactive metal ion delivery and teeth remineralisation

ABSTRACT

The invention provides, in one aspect, compositions for delivering a bioactive metal ion to a mammal, the compositions comprising (a) an effective amount of a source of the bioactive metal ion, (b) a phosphoprotein preparation obtained by partially cross linking a partial hydrolysate of casein or a caseinate, and (c) one or more physiologically acceptable diluents or carriers. Also provided are compositions for remineralising tooth enamel and/or for treating or preventing dental caries, tooth erosion, dentinal hypersensitivity or gingivitis in a mammal, wherein the compositions comprise an effective amount of such a phosphoprotein preparation, in combination with one or more carriers or diluents. In related aspects, the invention provides methods of using such compositions. Also provided are novel phosphoprotein preparations suitable for use in such compositions and methods.

FIELD OF THE INVENTION

This invention relates to compositions and methods for deliveringbioactive metal ions to humans and animals. It also particularly relatesto compositions and methods for remineralising teeth and/or forpreventing or treating dental caries and/or tooth erosion, dentinalhypersensitivity or gingivitis.

BACKGROUND OF THE INVENTION

Dental caries (or decay) and dental erosion are still widespreadconditions, despite the fluoridation of the water supply in manycountries and the use of fluoride toothpastes. Dental caries usuallybegins in the enamel of the tooth surface but may progressively destroythe hard tissues of the teeth. In many countries, about half of 5 yearold children experience some tooth decay. In addition, some groups ofpeople are, by virtue of their occupation, particularly susceptible todental erosion and/or caries. For example, wine tasters and athletessuch as elite cyclists who frequently sip on sports drinks, continuallyexpose their teeth to low pH beverages which may cause the whole surfaceof the tooth to dissolve.

It is well known that dairy products have a protective effect againstthe development of dental caries. A number of investigations havesuggested that it is primarily the protein component of dairy products,and casein in particular, that exerts an anticariogenic/remineralisingaction on tooth enamel. In addition, a particular fraction of activepeptides in casein have been identified as being largely responsible forthe anticariogenic/remineralising action. These are the calciumphosphate sequestering phosphopeptides, which constitute about 10% ofthe total weight of casein. These peptides contain a cluster ofphosphoseryl residues [-Ser(P)-Ser(P)-Ser(P)-Glu-Glu] that markedlyincrease the solubility of calcium phosphate by forming colloidal caseinphosphopeptide amorphous calcium phosphate complexes.

There are numerous patent publications directed to various compositionscontaining casein, caseinates, digests thereof or specificcasein-derived phosphopeptides for use in caries inhibition and relateddental applications.

For example, NZ patent specification 199891 describes toothpastes anddentifrices containing a caries and gingivitis inhibiting amount ofcasein, alpha-s-casein or phosvitin.

JP 59152317 describes an oral composition comprising mutanase (a drugfor preventing tooth decay) together with casein, casein hydrolysate ora mixture thereof.

U.S. Pat. No. 5,833,953, JP 9002928 and U.S. Pat. No. 5,427,769 alldescribe various compositions for treating or preventing dental cariesand containing micellar casein.

U.S. Pat. No. 5,130,123 describes a dentifrice composition forinhibiting caries or gingivitis containing a water soluble salt ofeither a caseinate or a digest of a caseinate.

WO 82/03008 describes compositions for inhibiting caries and gingivitis,containing phosphoproteins or phosphopolypeptides containing a specifiedamino acid sequence, and in particular sodium caseinate, calciumcaseinate or phosvitin.

JP 4077415 describes a dental calculus-preventing composition containingcasein phosphopeptides in combination with a suitable excipient.

U.S. Pat. No. 5,015,628 describes anticariogenic phosphopeptides having5 to 30 amino acids and containing a specified amino acid sequence, andwhich may be obtained by tryptic digestion of casein.

WO 98/40406 describes specific calcium phosphopeptide complexes havinganticaries efficacy.

The phosphopeptides contain the Ser(P) cluster sequence motif[-Ser(P)-Ser(P)-Ser(P)-Glu-Glu], and are said to be able to stabilizetheir own weight in amorphous calcium phosphate and amorphous calciumfluoride phosphate.

WO 00/06108 describes various formulations for the delivery of bioactiveconstituents to biological surfaces such as dental surfaces, comprisingsuspensions or solutions of one or more isolated and purified caseinprotein or salt thereof.

Compositions containing casein phosphopeptides have been reported ashaving superior anticaries/remineralising activity compared tocompositions containing intact casein. However, the use of caseinphosphopeptides has the disadvantage that digestion of casein (forexample using the enzyme trypsin) to release the desired phosphopeptidesalso releases hydrophobic peptides which give the resulting digest abitter flavour. This means that, for the product to have an acceptableflavour, fractionation of the digest to remove such hydrophobic peptidesis required. In turn, this means that only a fraction of thecasein-derived material is used; typically over 75% of the material iswasted.

The applicants have now surprisingly found that by partially hydrolyzingcasein and subsequently partially cross-linking the partial hydrolysate,phosphoprotein preparations having superior calcium-binding and teethremineralisation properties to those of unmodified casein or a partialcasein hydrolysate can be obtained. Such phosphoprotein preparationshave also been found to have an enhanced ability to bind other bioactiveactive metal ions.

JP 4-126039 describes a method of preparing a functional peptide bypartially hydrolyzing a food protein, such as a protein obtained fromsoy beans, wheat or sweetcorn, or animal proteins such as gelatin,animal meat, fish meat or casein, followed by treatment of the resultanthydrolyzed peptide with transglutaminase or diluted acid. The resultingtreated peptide is said to be free of bitterness. JP 4-126039 does nothowever describe the cation binding properties of such peptides, nor isa peptide obtained from casein specifically described therein.

WO 00/05972 and WO 01/0154512 describe chewing gum compositionscontaining casein or a modified casein such as polymerized hydrolyzedcasein, as part of the elastomeric component of the gum. Thesepublications do not however describe the cation binding properties ofsuch modified casein.

It is an object of the present invention to provide methods and/orcompositions useful for delivering bioactive metal ions, and methodsand/or compositions for remineralising teeth, and/or preventing ortreating dental caries, tooth erosion, dentinal hypersensitivity orgingivitis, which will go some way towards overcoming the disadvantagesof the prior art, or at least provide the public with a useful choice.

SUMMARY OF THE INVENTION

In a first aspect, the invention provides a composition for delivering abioactive metal ion to a mammal, the composition comprising an effectiveamount of a source of the metal ion, a phosphoprotein preparationobtained by partially cross linking a partial hydrolysate of casein or acaseinate, and one or more physiologically acceptable diluents orcarriers.

In another aspect, the present invention provides a method of deliveringa bioactive metal ion to a mammal, comprising administering to themammal a composition comprising an effective amount of a source of themetal ion in combination with a phosphoprotein preparation, wherein thephosphoprotein preparation has been obtained by partially cross linkinga partial hydrolysate of casein or a caseinate.

In a further aspect, the invention provides the use, in the preparationof a composition for delivering a bioactive metal ion to a mammal, of aphosphoprotein preparation obtained by partially cross linking a partialhydrolysate of casein or a caseinate.

Preferably, the metal ion is divalent.

In preferred embodiments, the metal ion is selected from the groupconsisting of calcium, iron, zinc, cobalt, copper and magnesium.

Preferably, the composition is an oral composition, in the form of afoodstuff or beverage, or a pharmaceutical vehicle such as a tablet orcapsule.

Preferably, the pH of the composition is between about 6 and 9, morepreferably between about 6.5 and 8, still more preferably between about6.8 and 7.7, and most preferably between about 7 and 7.5.

In a further aspect, the invention provides a composition forremineralising tooth enamel and/or for treating or preventing dentalcaries, tooth erosion, dentinal hypersensitivity or gingivitis in amammal, wherein the composition comprises an effective amount of aphosphoprotein preparation in combination with one or more carriers ordiluents, wherein the phosphoprotein preparation has been obtained bypartially cross linking a partial hydrolysate of casein or a caseinate.

In still a further aspect, the present invention provides a method forremineralising tooth enamel and/or for treating or preventing dentalcaries, tooth erosion, dentinal hypersensitivity or gingivitis in amammal, the method comprising contacting the teeth of the mammal with acomposition comprising an effective amount of a phosphoproteinpreparation, wherein the phosphoprotein preparation has been obtained bypartially cross linking a partial hydrolysate of casein or a caseinate.

In still a further aspect, the invention provides the use, in thepreparation of a composition for remineralising tooth enamel and/or fortreating or preventing dental caries, tooth erosion, dentinalhypersensitivity or gingivitis in a mammal, of a phosphoproteinpreparation obtained by partially cross linking a partial hydrolysate ofcasein or a caseinate.

In preferred embodiments, the composition further comprises a source ofcalcium ions.

Preferably, the composition also comprises a source of phosphate ions.

More preferably, the composition comprises both calcium and phosphateions, conveniently added as calcium phosphate. Alternatively, thecalcium and phosphate ions may be added as sodium phosphate and calciumchloride.

In a particular preferred embodiment, the source of calcium ionscomprises natural milk calcium, such as that available under the tradename ALAMIN®.

Preferably, calcium ions are present in the composition at a level of atleast about 5 mmol calcium ions per gram of phosphoprotein preparation,more preferably at least about 10 mmol/g, still more preferably at leastabout 20 mmol/g, more preferably at least about 30 mmol/g.

Preferably, the molar ratio of calcium ions to phosphate ions is in therange of about 0.8-1.2:0.4-0.8, more preferably about 1:0.6.

In another alternate embodiment, the composition comprises a source ofstrontium ions. Preferably, in this embodiment the composition alsocomprises a source of fluoride ions.

In certain preferred embodiments, the composition is in the form of afoodstuff such as cheese, conveniently a processed cheese, or aconfection, such as a chewing gum.

In alternative preferred embodiments, the composition is in the form ofa mouthwash or a dentifrice, such as a liquid dentifrice, a toothpaste,a powder, an emulsion or a gel.

Preferably the partial hydrolysate is obtained by enzymatic hydrolysisof acid casein, rennet casein or a caseinate.

Preferably, the enzyme is trypsin, conveniently bovine derived trypsinor porcine pancreatic trypsin.

Preferably, the partial hydrolysis is carried out at a pH of from about7 to about 8.

Preferably, the degree of hydrolysis is in the range of about 3% toabout 8%, more preferably about 3.5 to about 7%, such as about 4% toabout 6.5%, of the total number of peptide bonds.

Preferably, the degree of hydrolysis is such that about 10% or less,more preferably about 5% or less, of the casein or caseinate is renderedinsoluble at pH 7, by the partial hydrolysis.

Preferably, the partial hydrolysate is partially cross linkedenzymatically, using the enzyme transglutaminase, preferably at a pH offrom about 7 to about 8.

Preferably, the degree of partial cross lining is such that theresulting phosphoprotein preparation comprises about 10 μmol or morecross links per gram of protein, more preferably between about 10 andabout 250 mmol/g protein, more preferably between about 50 and 160μmol/g protein, such as between about 110 and 150 μmol/g protein.

In a further aspect, the present invention provides a novelphosphoprotein preparation, wherein the phosphoprotein preparation hasbeen obtained by partially cross linking a partial hydrolysate of caseinor a caseinate, and wherein the degree of partial hydrolysis of thecasein or caseinate prior to cross linking is in the range of about 3%to about 8%, more preferably about 3.5 to about 7%, such as about 4% toabout 6.5%, of the total number of peptide bonds, and the degree ofpartial cross linking is such that the phosphoprotein preparationcomprises about 10 μmol or more cross links per gram of protein, morepreferably between about 10 and about 250 μmol/g protein, morepreferably between about 50 and 160 mmol/g protein, such as betweenabout 110 and 150 μmol/g protein.

While the invention is broadly as defined above, it is not limitedthereto and also includes embodiments of which the following descriptionprovides examples.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in more detail and with reference tothe accompanying drawings, in which:

FIG. 1 shows the enamel remineralisation yield after treatment of acidetched tooth enamel with three different remineralising solutions,namely saliva, 10% caseinate solution, and 10% partially cross linkedhydrolyzed caseinate solution (as described in Example 3);

FIG. 2 is a Scanning Electron Micrograph of the tooth enamel surfaceprior to acid etching;

FIG. 3 is a Scanning Electron Micrograph of the tooth enamel surfaceafter acid etching;

FIG. 4 is a Scanning Electron Micrograph of the tooth enamel surfaceafter both acid etching and treatment with polymerized hydrolyzedcaseinate solution.

FIG. 5 shows the changes in microhardness of enamel after ademineralization treatment in 0.1 mol/L acetic acid pH 4.5 for 24 hoursfollowed by remineralisation in a MAP 112 phosphoprotein solutioncontaining 60 mmol/L calcium ions and 36 mmol/L phosphate ions;

FIG. 6 shows the changes in microhardness of enamel after ademineralization treatment in 0.1 mol/L acetic acid pH 4.5 for 48 hoursfollowed by remineralisation in a MAP 112 phosphoprotein solutioncontaining 60 mmol/L calcium ions and 36 mmol/L phosphate ions;

FIG. 7 shows the changes in microhardness of enamel after ademineralization treatment in 0.1 mol/L acetic acid pH 4.5 for 72 hoursfollowed by remineralisation in a MAP 112 phosphoprotein solutioncontaining 60 mmol/L calcium ions and 36 mmol/L phosphate ions;

FIG. 8 shows the changes in microhardness of enamel after ademineralization treatment in 0.1 mol/L acetic acid pH 4.8 for 48 hoursfollowed by remineralisation in a MAP 112 phosphoprotein solutioncontaining 60 mmol/L calcium ions and 36 mmol/L phosphate ions;

FIG. 9 shows the changes in microhardness of enamel after ademineralization treatment in 0.1 mol/L acetic acid pH 4.5 for 24 hoursfollowed by remineralisation in a MAP 112 phosphoprotein solutioncontaining 60 mmol/L calcium ions and 36 mmol/L phosphate ions; and

FIG. 10 shows the changes in microhardness of enamel after a caries-likedemineralisation treatment followed by remineralisation in a MAP 112solution containing 500 mg/mL hydroxyapatite and varying levels calciumand phosphate added as soluble salts;

FIG. 11 is an image of a demineralised tooth enamel taken using a ZeissUpright Confocal Microscope. Enamel was demineralised for 96 hours in apH 4.8 buffer;

FIG. 12 is an image of a Zeiss Confocal Microscope image ofdemineralised tooth enamel that was remineralised in MAP112 proteincontaining 75 mM calcium chloride and 45 mM sodium phosphate buffer. Theexposed hexagonal microstructure has been remineralised extensivelyleaving calcium deposits on the enamel surface.

FIG. 13 shows the effect of MAP phosphoprotein concentration on thepercent of C. albicans inhibited from adhering to hydroxyapatite beadsover three experiments;

FIG. 14 shows the effect of MAP phosphoprotein concentration on thepercent of S. mutans inhibited from adhering to hydroxyapatite beadsover three experiments. It can be seen that as the concentrationincreases the % inhibition of adhesion increases; and

FIG. 15 shows the changes in microhardness of enamel after a caries-likedemineralization treatment followed by remineralisation in a processedcheese (containing phosphoprotein MAP 112 and milk calcium phosphate)extract solution.

FIG. 16 is an image of a demineralised tooth enamel taken using a ZeissUpright Confocal Microscope. Enamel was demineralised for 96 hours in pH4.8 buffer;

FIG. 17 is an image of a Zeiss Confocal Microscope image ofdemineralised tooth enamel that was remineralised in cheese extract. Theenamel has been remineralised extensively filling in the areas betweenthe hexagonal microstructure.

DESCRIPTION OF THE INVENTION

As defined above, the present invention relates to methods andcompositions for delivering bioactive metal ions to mammals. It alsorelates to methods and compositions for remineralising tooth enameland/or for treating or preventing dental caries, tooth erosion, dentinalhypersensitivity or gingivitis. In a related aspect, the invention alsorelates to novel phosphoprotein preparations useful in such methods andcompositions.

The applicants have surprisingly found that by partially hydrolyzingcasein or a caseinate and subsequently partially cross linking thepartial hydrolysate, the resulting phosphoproteins have both anincreased solubility in the presence of divalent cations and anincreased ability to bind divalent cations when compared to the noncross-linked partially hydrolysed casein.

These properties make such phosphoprotein preparations useful asdelivery vehicles for administering bioactive metal ions to humans oranimals, particularly divalent cations including calcium, iron, zinc,cobalt, copper and magnesium. The phosphoprotein preparations are ableto bind significant quantities of cations and thus prevent precipitationof and maintain salts of the cations in solution, thereby enhancingtheir bioavailability. Also, as the phosphoprotein preparations havebeen found to have a higher solubility than unmodified casein, they canbe incorporated into oral compositions relatively easily.

The phosphoprotein preparations have particular application inremineralising tooth enamel and thereby in treating or preventing tootherosion or dental caries. Without wishing to be bound by any theory, itis believed that the ability of the phosphoprotein preparations tosolubilise calcium (and phosphate) ions may be at least partlyresponsible for their ability to remineralise tooth enamel. Inparticular, the phosphoprotein preparations are able to maintain a highconcentration of calcium and phosphate ions in solution close to thetooth enamel surface, facilitating diffusion of these ions back into thepores of demineralised tooth enamel and thus increasing remineralisationactivity. The phosphoprotein preparations have also been found to form acoating on the tooth enamel surface, which may further enhance theavailability of calcium and phosphate ions.

Moreover, the applicants have also surprisingly found that thephosphoprotein preparations inhibit the adhesion of caries-causingbacteria, in particular Streptococcus mutans to the enamel surface. Asthe bacteria need to adhere to the tooth surface to initiate the cariesprocess, reducing the degree of adhesion of the bacteria can reduce therisk of caries formation. Accordingly, the phosphoprotein preparationsof the present invention possess not only a reparative, remineralisationfunction, but also a preventative function. The applicants have alsofound that the protein can inhibit the adhesion of the yeast C.albicans, which is involved in oral thrush.

The phosphoprotein preparations suitable for use in the invention may beobtained by partially cross linking a partial hydrolysate of casein,that is, casein in which some, but not all, of the peptide bonds havebeen hydrolyzed.

The casein used to prepare the partial hydrolysate may be in any form;acid casein, rennet casein or a caseinate may all be used. Althoughchemical hydrolysis is by no means excluded, it is preferred that thepartial hydrolysis is carried out enzymatically, in aqueous solution.Suitable enzymes for performing the partial hydrolysis include proteasessuch as trypsin and chymotrypsin. It is however particularly preferredthat that the enzyme used is trypsin, conveniently bovine derivedtrypsin or porcine pancreatic trypsin.

The partial hydrolysis may be carried out at a temperature and pHappropriate to the enzyme being used. For example, if bovine derivedtrypsin is used, the partial hydrolysis may conveniently be carried outat a pH of about 7 to about 8, and at a temperature of about 37° C. Itwill be appreciated that at this pH, the casein will be present as acaseinate, eg sodium caseinate, depending on the buffer used in thereaction solution.

The reaction should be carried out for a sufficient period of time andunder appropriate conditions, eg enzyme and casein concentrations, toallow the desired degree of hydrolysis to be achieved. When the desireddegree of hydrolysis has been achieved, the reaction may conveniently beterminated, or at least substantially terminated, by inactivating theenzyme, for example by heating the reaction mixture to a temperaturewhich will denature the enzyme, eg about 80° C. It is not critical thatthe hydrolysis reaction be completely terminated, provided the partialcross linking reaction as discussed below is commenced once the desireddegree of hydrolysis has been achieved. That is, a minor amount ofhydrolysis may still continue while the partial cross linking reactionis being carried out.

It is preferred that the partial hydrolysis is carried out underconditions which result in the partially hydrolyzed casein having adegree of hydrolysis in the range of about 3% to about 8%, morepreferably about 3.5% to about 7%, such as about 4% to about 6.5%, interms of the percentage of the original peptide bonds hydrolysed.

It is also preferred that the degree of hydrolysis is such that about10% or less, more preferably about 5% or less, of the casein orcaseinate is rendered insoluble at pH 7, by the partial hydrolysisprocess.

The degree of hydrolysis may be measured by methods known to thoseskilled in the art, conveniently by the TNBS (2, 4, 6-trinitrobenzenesulfonic acid) method.

Preferably, the molecular weight profile of the partially hydrolyzedcasein or caseinate is less than that of casein but greater than thefollowing distribution: about 1.7%≧30,000 Da, 22%<30,000 Da and ≧21,000Da, 22%<21,000 Da and ≧12,000 Da, 54.3%<12,000 Da.

More preferably, the molecular weight profile of the partiallyhydrolyzed casein or caseinate is less than that of casein but greaterthan the following distribution: about 9.4%≧30,000 Da, 48%<30,000 Da and≧21,000 Da, 11%<21,000 Da and ≧12,000 Da, 31.6%<12,000 Da.

More preferably, the molecular weight profile of the partiallyhydrolyzed casein or caseinate is less than that of casein but greaterthan the following distribution: about 11%≧30,000 Da, 50%<30,000 Da and≧21,000 Da, 10%<21,000 Da and ≧12,000 Da, 29%<12,000 Da.

Most preferably, the molecular weight profile of the partiallyhydrolyzed casein or caseinate is less than that of casein but greaterthan the following distribution: about 13%≧30,000 Da, 53%<30,000 Da and≧21,000 Da, 8%<21,000 Da and ≧12,000 Da, 26%<12,000 Da.

The molecular weight profile of the partially hydrolyzed casein mayconveniently be measured by size exclusion gel filtration, usingtechniques known to those persons skilled in the art. By way of example,the partially hydrolyzed casein may be dissolved in a suitable solvent,conveniently 6M urea, and the protein fractions separated using fastprotein liquid chromatography (FPLC system), for example using aSuperdex 200 10/30 HR column, and the eluted proteins detected by UVabsorption (conveniently at 280 nm). The molecular weight distributionof the eluted proteins may then be calculated by integration of theprotein absorption curve. Those skilled in the art will appreciate thatthe protein absorption curve will be dependent on the choice of bufferand buffer concentration.

Those persons skilled in the art will appreciate that by varying thereaction conditions appropriately, such as the reaction time and enzymeconcentration, a partially hydrolyzed casein having the desired degreeof hydrolysis can be obtained. By way of example, a partially hydrolyzedcasein having a suitable degree of hydrolysis may be obtained by firstsolubilising a 10% isoelectric precipitated casein solution with NaOH topH 7 at 50° C. The solution is then cooled to 37° C., and a porcinepancreatic trypsin preparation (a suitable preparation is commerciallyavailable as Novo.4500 K, molecular weight 23,400 Da, activity 4500 USPunits/mg) added at about 0.01% w/w casein and incubated for 15 minutes.Enzyme inactivation may be achieved by heating to 80° C. and holding for5 minutes.

Once the partially hydrolyzed casein has been prepared, it is thenpartially cross linked to form a phosphoprotein preparation according tothe invention.

As used herein, the term “cross linking”, when used in the context ofpartially cross linking a partial hydrolysate of casein or a caseinate,means the formation of intermolecular covalent bonds between the aminoacid residues of the casein molecules and/or casein molecule fragmentscomprising the partial hydrolysate. Preferably, the intermolecularcovalent bonds comprise bonds between glutamine and lysine residues, ieglutamyl/lysyl covalent bonds. It will also be appreciated that someintramolecular cross linking, ie between amino acid residues on the samecasein molecule or casein fragment, is likely to occur.

The term “partial”, when used in the context of “partial cross linking”,means that not all of the amino acid residues are cross-linked, ie thatsome non-cross linked amino acid residues will remain following thecross linking reaction.

The degree of partial cross linking is expressed herein in terms ofmicromoles of cross links per gram of protein.

The degree of partial cross linking, in terms of the quantity ofglutamyl/lysl bonds, may conveniently be determined by high performanceliquid chromatography (HPLC), by carrying out a proteolytic digestion ofthe cross-linked proteins using suitable enzymes, conveniently pronase,leucine aminopeptidase, prolidase and carboxypeptidase, followed by HPLCof the proteolytic digest and quantification of the ε-(γ-Glutamyl)lysine(G-L) peak.

The partial cross ling may conveniently be carried out enzymatically,using either of the enzymes lysyl oxidase or transglutaminase.

It is particularly preferred that the enzyme transglutaminase is used,and that the polymerization is carried out at a pH between about 7 and8. The partial cross linking is desirably carried out under conditionsand for a time sufficient to allow the desired degree of cross linkingto take place. It is preferred that the reaction be carried out underconditions such that the degree of cross linking in the resultingphosphoprotein preparation comprises about 10 μmol or more cross linksper gram of protein, more preferably between about 10 and about 250μmol/g protein, more preferably between about 50 and 160 μmol/g protein,such as between about 110 and 150 μmol/g protein.

Again, once the desired degree of cross linking has been achieved, thereaction can be terminated by inactivation of the enzyme, typically byheating the reaction mixture to a temperature sufficient to denature theenzyme, for example to about 80° C. for about 5 minutes. It is alsogenerally preferred that, following completion of the partial crosslinking and deactivation of the enzyme, the resultingphosphoprotein-containing solution is dialyzed or diafiltered to removeany remaining low molecular weight peptides and salts, convenientlyusing a membrane with a molecular weight cutoff of from about 10,000 toabout 14,000 Da. The purified phosphoprotein-containing solution may befreeze dried or spray dried to obtain the phosphoprotein preparation ina solid form.

Any commercially available source of transglutaminase can be used tocarry out the partial cross linking. By way of example, a suitableenzyme is a 1% transglutaminase preparation commercially available fromAjinomoto Co. as Activa MP.

Alternatively, the plastein reaction (which is an enzymatic reactionknown to those skilled in the art) may be used. The cross linking mayalso be carried out between tyrosine residues using peroxidase andhydrogen peroxide.

Although it is preferred that partial cross linking of the partialcasein hydrolysate is carried out enzymatically, partial cross linkingby chemical means, using a suitable reagent such as a bifunctionalaldehyde (eg glutaraldehyde) is not excluded.

Those persons skilled in the art will appreciate that by varying thereaction conditions appropriately, such as the reaction time and enzymeconcentration, a phosphoprotein preparation having the desired degree ofcross linking can be obtained.

By way of example, a phosphoprotein preparation having a suitable degreeof cross linking may be obtained by treating a partially hydrolyzedcasein prepared as described above with a transglutaminase preparation(Activa MP, commercially available from Ajinomoto Co.) added at a ratioof 4.5% w/w casein and incubating the reaction mixture at 40° C. for 18hours.

The phosphoprotein preparations may be incorporated into compositionssuitable for delivering bioactive metal ions, particularly divalentmetal ions, to humans and animals. Such compositions may be in the formof pharmaceutical vehicles such as tablets or capsules. Tablets orcapsules containing the phosphoprotein preparation, in combination witha source of a bioactive metal ion in a physiologically useful amount andone or more physiologically acceptable carriers or diluents, may beprepared using standard methods known to those skilled in the art. Thedesired quantities, ie effective amounts, of the source of bioactivemetal ion to be incorporated in the compositions of the presentinvention will vary depending on the particular cation in question andthe amount in which it is required by the mammal for whom thecomposition is intended, for example whether it is a trace mineral suchas iron, zinc, manganese, molybdenum, copper, chromium, or is requiredin larger amounts, such as calcium.

Alternatively, the phosphoprotein preparation may be incorporated into afoodstuff or beverage, in combination with a source of the bioactivemetal ion it is desired to administer. For example, compositionscontaining the phosphoprotein preparations in combination with aneffective amount of a source of calcium (such as calcium phosphate) oriron may be administered to humans or animals in need of calcium or ironsupplementation, respectively.

The phosphoprotein preparations also have particular application inremineralising teeth, and in treating or preventing tooth erosion,dental caries, dentinal hypersensitivity or gingivitis.

Compositions useful in such applications and suitable for contactingteeth with the phosphoprotein preparation may take a number of forms.For example, such compositions may take the form of a mouthwash or adentifrice, such as a liquid dentifrice, toothpaste, a powder, anemulsion or a gel containing the phosphoprotein. Alternatively, thephosphoprotein preparations may be incorporated into foodstuffs, such ascheese, for example processed cheese, or confectionery such as chewinggum.

It is preferred that, in addition to the phosphoprotein preparation,such compositions also contain a source of calcium ions, and preferablyalso a source of phosphate ions. For example, calcium phosphate may beincluded in the composition. Although calcium and phosphate ions arepresent in saliva, incorporating sources of these ions is preferred inorder to take advantage of the calcium-binding properties of thephosphoprotein and maximize the concentration of calcium and phosphateions in contact with the tooth enamel.

It is particularly preferred that calcium ions are present in thecomposition at a level of at least about 5 mmol calcium ions per gram ofphosphoprotein preparation, such as at least about 10 mmol/g, such asabout 20 mmol/g, such as about 30 mmol/g. It is also preferred that themolar ratio of calcium ions to phosphate ions is in the range of about0.8-1.2:0.4-0.8, more preferably about 1:0.6.

In some particularly preferred embodiments, the source of calcium ionscomprises natural milk calcium phosphate, in which the calcium isgenerally in the form of calcium hydroxyapatite.

Natural milk calcium phosphate is commercially available, with aparticularly preferred embodiment being that available from New ZealandMilk Products Ltd under the trade name ALAMIN®, which comprises calcium,phosphate, and also protein, lactose, fat, moisture, sodium, potassiumand chloride, in the following typical proportions: Total mineralcontent 70% (w/w): 28% calcium and 48% phosphate Protein (N × 6.38) 7%Lactose 4% Fat 1% Free moisture 3% Bound moisture 8% Calcium 28,000mg/100 g Phosphorus 16,000 mg/100 g Sodium 400 mg/100 g Potassium 300mg/100 g Chloride 100 mg/100 g

Natural milk calcium phosphate may be obtained by methods known in theart, typically by clarifying and pasteurizing acid whey permeate,cooling and ultrafiltering the permeate, followed by heating, pHadjustment and holding at the elevated temperature such that theminerals including calcium phosphate will be precipitated, and recoveryof the precipitate.

Another suitable source of natural milk calcium phosphate is the productavailable under the trade mark Lactoval® from DMV International.

Other anions that may be included in the compositions include fluorideand fluorophosphate.

In other embodiments, the compositions may include a source of strontiumions in addition to or instead of calcium ions. Such compositions may beparticularly useful in treating dentinal hypersensitivity.

It is generally preferred that the pH of the compositions of the presentinvention, either in the form of compositions for delivering bioactivemetal ions or for dental applications as described above, is buffered ata level between about 6 and 9, more preferably between about 6.5 and 8,still more preferably between about 6.8 and 7.7, and most preferablybetween about 7 and 7.5.

In one embodiment of the invention, a processed cheese product isprovided, which includes a phosphoprotein preparation as describedabove, in combination with natural milk calcium phosphate. In thisembodiment of the invention, it will be appreciated that the productacts both as a source of dietary calcium as well as havingremineralising/anticaries properties.

The processed cheese component of the product is preferably present inan amount of about 90 to about 98% by weight of the product, such asabout 94 to about 96% by weight, typically about 95% by weight Thephosphoprotein preparation may typically comprise about 0.5 to about 3%by weight of the product, such as about 1 to about 2%, typically about1.2 to about 1.6% by weight. The natural milk calcium phosphate maytypically comprise about 0.5% to about 4.5% by weight of the product,such as about 2 to about 3.5%, typically about 2.5% to about 3% byweight.

In another specific embodiment of the invention, a composition in theform of an emulsion is provided, comprising a phosphoprotein preparationas described herein in combination with natural milk calcium,conveniently that commercially available as ALAMIN®. The phosphoproteinpreparation may conveniently be present in an amount of about 1% toabout 15% by weight of the emulsion, such as about 5% to about 12%,typically about 9% to 11%. The natural milk calcium phosphate may bepresent in an amount of about 3% to about 12% by weight of the emulsion,such as about 5% to about 10%, typically about 6 to about 9% by weight.The emulsion will preferably include one or more additional components,such as emulsifiers, thickeners, flavourings and sweeteners. Suchadditional components may be chosen from those known in the art assuitable for use in emulsion type formulations for dental use.

The invention will now be described in more detail with reference to thefollowing non-limiting examples.

EXAMPLES Example 1

Phosphoprotein Preparation and Calcium Binding

Tryptic Hydrolysis

A 10% isoelectric precipitated casein solution was solubilised with NaOHto pH 7.0 at 50° C. Once soluble, the solution was cooled to 37° C., andbovine derived trypsin (Novo) added at between 0.01-0.2% w/w casein,incubated for up to 2 hours, then heated to 80° C. and held for 5minutes. Enzyme concentration Hydrolysis time/ Degree of Lot number %weight/weight casein minutes Hydrolysis MAP10 Lot 5 0.01 15 5.7 Lot 60.01 30 6.0 Lot 7 0.01 45 6.3 Lot 1 0.01 60 7.0

The molecular weight profiles of the phosphoprotein preparations weredetermined by gel filtration as follows. A 1% protein solution wasprepared in 6M Urea, with 50 mM sodium phosphate at pH 7.5 as thebuffer. This solution was centrifuged at 10 000×g for 10 minutes andpassed through a 0.2 μm filter. A sample volume of 500 μl injected intothe 100 μl sample loop of a Pharmacia FPLC fitted with a Superdex 20010/30HR column. The running buffer used was 6 M Urea, with 50 mM sodiumphosphate at pH 7.5 and flow rate of 0.5 ml/min. Detection was by UVabsorption (280 ηm). The protein absorption curve was integrated andarbitrarily divided into the following four molecular weight groupings:

1) greater than about 30,000 Daltons

2) less than about 30,000 Daltons and greater than about 21,000 Daltons

3) less than about 21,000 Daltons and greater than about 12,000 Daltons

4) less than about 12,000 Daltons.

Molecular Weight Profiles of Lots 5, 6, 7 and 1 (After PartialHydrolysis), Expressed as % Distribution of Lots 5, 6, and 1 Lot numberMolecular weight range 5 6 7 1 ≧30,000 13.66 11.66 9.4 9.84 <30,000,≧21,000 53.74 50.78 49.13 48.87 <21,000, ≧12,000 7.58 9.99 11.37 10.92<12,000 25.02 27.57 30.1 30.37Transglutaminase Treatment

The pH was re-adjusted (as necessary) to 7.0, and transglutaminase (1%commercial preparation, Ajinomoto) added at a ratio of 4.5% w/w caseinand incubated at 40° C. for the desired length of time. Enzymeinactivation was achieved by heating to 80° C., and holding for 5 min.The modified protein solutions were freeze dried.

Number of Cross Links Formed after Treatment with Transglutaminase,Expressed as μmol Cross-Link/G Protein for Lots 5, 6, 7 and 1Cross-linking time (hours) Lot number 1 6 18 Lot 5 50 98 168 Lot 6 51 90152 Lot 7 55 95 150 Lot 1 57 109 159Measurement of Calcium Binding Capacity of the PhosphoproteinPreparations

The calcium binding capacity of the proteins was determined byre-suspending the protein in water, adding calcium and phosphate ions ata set ratio under constant pH; removing the insoluble material (saltsand protein); then removing the soluble non bound salts and determiningthe amount of calcium bound to the soluble protein. The experimentaldetails were as follows.

A 1% solution of the proteins were dissolved with milli-Q water, andallowed to stand for 1 hour to ensure complete hydration. Calciumchloride was added at the following levels: 0 mM, 10 mM, 20 mM, 30 mM,40 mM and 50 mM; and the solution incubated at 25° C. for 1 hour withgood mixing. Sodium phosphate was added at a molar ratio of 0.6 to thecalcium. Throughout the experiment the pH was maintained at 7.0 usingNaOH solution. The samples were incubated at 25° C. for 6 to 10 hourswith good mixing. After incubation, a sample was centrifuged at 10 000×gfor 10 minutes and filtered through a 0.2 μm nylon filter and split intotwo portions.

One portion of the sample was injected into a 2 ml sample loop andloaded onto a Pharmacia FPLC fitted with Sephadex G-25 (Vt=25 ml)desalting column. The running buffer was 10 mM HEPES at pH 7 the flowrate was 2 ml/min and detection was achieved through UV absorption (280em), conductivity and pH. The protein peak was collected and calciumconcentration determined by atomic absorption spectroscopy (AAS).

The other portion was used to determine the soluble protein content asper the Folin protein assay.

Calcium Binding Capacity of Lot 6 before (0 hours) and afterTransglutaminase Treatment (1, 6, or 18 hours) Expressed as mg Ca²⁺ perg Initial Protein mmol Ca²⁺ Hours treated with transglutaminase added 01 6 18 30 3.2 7.3 24.2 31.2 40 1.1 1.2 34.7 37.6

Calcium Binding Capacity of Lot 6 before (0 hours) and afterTransglutaminase Treatment (1, 6, or 18 hours) Expressed as mg Ca²⁺ perg Initial Protein mmol Ca²⁺ Hours treated with transglutaminase added 01 6 18 30 9.3 32.3 32.1 40.2 40 0.0 2.6 7.1 4.2

Calcium Binding Capacity of Lot 7 before (0 hours) and afterTransglutaminase Treatment (1, 6, or 18 hours) Expressed as mg Ca²⁺ perg Initial Protein mmol Ca²⁺ Hours treated with transglutaminase added 01 6 18 30 16.1 24.6 28.5 32.0 40 3.0 2.4 n/a 7.4

Calcium Binding Capacity of Lot 1 before (O hours) and afterTransglutaminase Treatment (1, 6, or 18 hours) Expressed as mg Ca²⁺ perg Initial Protein mmol Ca²⁺ Hours treated with transglutaminase added 01 6 18 30 24.6 16.0 21.9 36.4 40 2.4 1.9 6.1 21.1

Calcium Binding Capacity of Caseinate Expressed as mg Ca²⁺ per g InitialProtein mmol Ca²⁺ added mg Ca²⁺ bound 0 0.0 5 2.8 10 2.3 15 1.7The different proteins described above represent a range of degrees ofhydrolysis of casein (Lot 5 the least hydrolysed, Lot 1 the most), and arange of cross-linking of the hydrolysis products with transglutaminase(0 hours, no cross-linking and 18 hours the most cross linked). Lot 5proteins are the most resistant to Ca²⁺ induced precipitation with nearmaximum calcium loading 40 mg Ca/g protein still remaining soluble in a40 mM Ca²⁺ solution.

With a greater degree of hydrolysis of the casein, the proteins, evenafter extensive Tg cross-linking, become less resistant to precipitationin the presence of higher Ca²⁺ concentrations. None of the non-crosslinked samples were resistant to precipitation, and resistance increasedwith greater cross-linking in all samples.

The ability of native casein to bind Ca²⁺ is demonstrated in the finaltable. It is rapidly precipitated with increasing Ca²⁺ concentrationsand has a maximum binding of just 2.8 mg Ca²⁺/g casein at 5 mM Ca²⁺.

The caseinate results illustrate the surprising nature of the partiallycross-linked hydrolysed casein solubility/binding results that proteinswith an average molecular weight similar to unmodified caseinate (orgreater than caseinate) should bind substantially more calcium ions andremain soluble.

Example 2

Preparation of a Phosphoprotein

Tryptic Hydrolysis

A 10% isoelectric precipitated casein solution was solubilised with NaOHto pH 7.0 at 50° C.

Once soluble, the solution was cooled to 37° C., and porcine derivedtrypsin (Novo.4500K, molecular weight 23,400 Da, activity 4500 USPunits/mg) added at 0.01% w/w casein and incubated for 15 minutes. Enzymeinactivation was achieved by heating to 80° C., and holding for 5minutes.

Molecular weight profiles of Lot 5 Molecular weight range ≧30,000 13.66<30,000, ≧21,000 53.74 <21,000, ≧12,000 7.58 <12,000 25.02Transglutaminase Treatment

The pH was re-adjusted (as necessary) to 7.0, and transglutaminase (1%commercial preparation, Activa MP, Ajinomoto Co) added at a ratio of4.5% w/w casein and incubated at 40° C. for 18 hours. Enzymeinactivation was achieved by heating to 80° C., and holding for 5 min.The molecular weight material greater than 30,000 Da was increased by100%.

Example 3

Teeth Remineralisation (Hardening)

The remineralising (rehardening) potential of three products wasdetermined. The products were human saliva, caseinate solution and aphosphoprotein preparation obtained by partial cross linking ofpartially hydrolyzed casein. The remineralising efficacy of the productswas determined by measuring the recovery in hardness of the controlledacid etched human enamel following treatment.

Enamel Preparation

The human unerupted third molars were used in all of the experiments.After extraction, the teeth were mechanically cleaned with anylac brushand deionised water, and stored until required in 10% buffered formailinsolution (pH 7.0) at 4° C.

Before use, the teeth were thoroughly rinsed, cut longitudinally andthen were embedded in epoxy resin (Araldite). Each specimen washand-ground on a glass plate using silicon carbide grits, progressivelyof 240- to 600-grit, under running water. Fine polishing was achievedusing the 8-inch Laps with 3-μm diamond abrasive for 5 minutes and with1-μm diamond abrasive for another 5 minutes on a rotating polishingmachine using distilled water to keep specimens moist. Between eachpolishing treatment, an ultrasonic bath was used for removing debris.The specimens were evaluated under a dissecting microscope (15×) andthose with any evidence of cracks, flaws, developmental defects orextraction damage were rejected. The selected samples were stored untilrequired in 10% buffered formalin solution (pH 7.0) at 4° C.

Enamel Demineralisation

The polished sound enamel specimens were individually demineralised in25 ml of 1% (w/v) citric acid solution for 10 minutes at 37° C. tocreate eroded lesions. Following demineralisation, specimens were washedthoroughly with deionised water and stored in deionised water prior tothe next step. The pH of the solution was 2.3.

Remineralisation

Preparation of the Remineralisation Products

Saliva: Submandibular saliva was collected from a healthy volunteer and10 mmol/L sodium azide added. Twenty-five millilitres of saliva was usedfor each specimen in the remineralising procedure.

10% Caseinate Solution (CN-60Ca): CN-60Ca contained 36 mmol/L (PO₄)³⁻(at pH 9.0), 60 mmol/L Ca²⁺ and 10% (w/v) lactic acid casein. Thesolution was thoroughly mixed using a magnetic stirrer at roomtemperature. After incubation in a water bath at 50° C. for 15 minutesand cooling to room temperature, 10 mmol/L sodium azide was added as apreservative and then adjusted to pH 7.5 with 10% (w/v) NaOH.

10% Phosphoprotein Solution (PC-60Ca): PC-60Ca consisted of 36 mmol/L(PO₄)³⁻ (at pH 9.0), 60 mmol/L Ca²⁺ and 10% (w/v) phosphoproteinpreparation obtained by partial cross linking of partially hydrolyzedcaseinate (PC). The phosphoprotein preparation was obtained by treatinga caseinate solution having a degree of hydrolysis of approximately 4%with the enzyme transglutaminase, as described in Example 2. Otherconditions were as described for CN-60Ca7.5.

Protocol for Enamel Remineralisation

The selected specimens were treated in separate remineralisationsolutions. The specimens were immersed in 40 ml of freshremineralisation solution (under constant shaking) for four hour timeperiods at 37° C. in individual sealed 80-ml beakers. The CN60Ca wasalso incubated for a 16 hour time period.

Evaluation of Demineralisation/Remineralisation

Surface Microhardness (SMH) Evaluation

The surface microhardness (SMH) of the enamel blocks were measured witha Leitz MiniLoad-Hardness instrument using a Vickers diamond under a 200g load for 20 seconds. The specimens were held perpendicular to theindentor to the compound stage of the hardness instrument. Fifteenindentations were averaged on each surface of the individual specimensfor surface hardness determinations (after standardisation of thediamond indenter and using the 400× magnification). A distance of atleast two times the indentation lengths for enamel was kept between theindentations to minimise interactions between neighbouring indentations.The SMH readings were taken at three stages of enameldemineralisation/remineralisation model as follows: (1) before exposureto the demineralisation solution (DS), (2) after exposure to the DS (1%citric acid) and softening and (3) after exposure to remineralisationsolution or control solution. Following the initial examination of SMH(stage 1), each specimen underwent 10 minutes of demineralisation at 37°C. in DS. Each specimen was immersed individually in 25 ml of solution.After exposure, the specimens were then removed from the solution,washed thoroughly in deionised water, blotted dry with 3 mm filter paperand taken for SMH re-examination (stage 2). After the selectedremineralisation treatment, the specimens were removed from thesolution, thoroughly washed in deionised water, blotted dry again andre-tested for SMH (stage 3). The softening and rehardening potentials ofthe DS and the different remineralisation solutions were calculated foreach specimen by subtracting the baseline SMH value (stage 1) andrehardening value (stage 3), respectively, from the softening value(stages 2).

The relationship between the measured SMH and the length of the Vickersindent average of the two diagonals was determined by equation 1:SMH (kg/mm2)=1854×P/d2 [1]where P is the load in grams and d is the average length of thediagonals of the indentation measured in microns. The SMH numbers aredirectly from the microscopic measurements with Leitz MiniLoad-Hardnessinstrument used in the present study. In order to compare quantitativelythe SMH numbers for the de- and remineralised specimens on a linearscale, it was necessary to convert the pre- and post-SMH measurements tovalues proportional to the yield of change between softened SMH and theamount of remineralisation achieved. For comparison between these tworesults, the difference of the remineralising effect was calculatedusing equation 2:R(%)=[(PSMH)3−(PSMH)2]/[100−(PSMH)2]×100%  [2]where PSMH3 and PSMH2 mean as follows:PSMH3=[SMH of stage 3/SMH of stage 1]×100%PSMH2=[SMH of stage 2/SMH of stage 1]×100%Therefore R in equation 2 is the rehardening yield, remineralisationyield, or recovery yield, of stage 3 in this study.

Results TABLE 1 Effect of submandibular saliva, caseinate solution andphosphoprotein preparation (obtained by partial cross linking ofpartially hydrolysed casein) on the etched enamel surface SurfaceMicrohardness (kg/mm²) Stage 1 Stage 2* Stage 3 Specimen No Mean (SD)Mean (SD) Mean (SD) Saliva for 4 hours: Saliva 1 398.6 (21.8) 331.4(34.3) 380.3 (30.5) ** Saliva 2 370.3 (13.6) 285.0 (35.3) 349.7 (22.3)** Saliva 3 405.0 (27.7) 290.3 (26.4) 352.6 (20.8) ** Saliva 4 379.6(14.9) 293.2 (25.6) 344.3 (17.8) ** CN-60Ca for 4 hours: CN 4 350.6(30.4) 281.9 (15.4) 279.1 (20.3) *** CN 5 405.9 (29.5) 319.7 (20.3)314.0 (23.2) *** CN 6 370.4 (19.1) 282.3 (26.7) 293.0 (26.6) *** CN 7397.2 (18.6) 313.4 (19.5) 316.0 (15.8) *** CN-60Ca for 16 hours: CN 8394.5 (19.0) 293.4 (33.3) 339.1 (26.4) ** CN 9 422.5 (24.5) 339.8 (23.7)325.1 (20.1) *** CN 10 376.5 (30.4) 324.2 (30.7) 362.4 (30.5) (p =0.003) CN 11 387.0 (20.4) 315.8 (30.0) 335.6 (20.5) (p = 0.015) PC-60Cafor 4 hours PC-60Ca 1 356.9 (20.5) 307.0 (23.2) 338.5 (36.9) ** PC-60Ca2 411.7 (19.5) 296.9 (22.5) 336.7 (35.4) ** PC-60Ca 3 378.0 (20.4) 300.4(15.1) 384.9 (16.1) ** PC-60Ca 4 374.9 (14.7) 281.2 (19.5) 394.9 (25.0)** PC-60Ca 5 379.6 (13.9) 286.6 (19.5) 400.3 (28.9) ** PC-60Ca 6 365.2(11.8) 293.5 (26.3) 364.3 (18.3) ** PC-60Ca 7 374.9 (30.3) 324.4 (30.5)364.9 (30.6) ** PC-60Ca 8 405.1 (21.3) 336.7 (19.8) 406.5 (37.1) **PC-60Ca 9 388.1 (21.9) 280.0 (36.2) 396.7 (22.8) ** PC-60Ca 10 399.5(26.3) 289.8 (09.1) 369.3 (17.0) ** Control groups: PC-noCa 1 347.6(19.2) 269.4 (13.7) 277.4 (20.5) *** PC-noCa 2 367.7 (18.7) 302.4 (20.6)307.5 (25.7) *** noPC-60Ca 1 362.5 (18.9) 299.4 (20.6) 278.7 (18.7) ***noPC-Ca 2 373.0 (21.0) 294.1 (22.2) 291.0 (31.4) ***N = 15 (each specimen).Stage 1: Microhardness testing in initial stage.Stage 2: Microhardness testing after 1.0% citric acid etching for 10minutes.Stage 3: Treatment group: Microhardness testing after treatment withremineralising solution.*All stage 2 values were significantly less than stage 1 values (P <0.001).** Stage 3 values were significantly greater than stage 2 values intreatment group (P < 0.001).*** Stage 3 values were not significantly greater than stage 2 values incontrol group.The Effect of Saliva on Etched Enamel Surface

Table 1 shows that human submandibular saliva significantly increasedthe SMH numbers of etched enamel after the four-hour treatment. Therelative SMH after acid etching (Stage 2) was decreased to 77.3% of itsoriginal value and after treatment with the saliva this increased to91.9% (Stage 3). The mean of the remineralisation yield (R) was 65.5%.

The Effect of 10% Caseinate (CN) on Etched Enamel Surface

It required 16 hours treatment with 10% caseinate solution (CN-60Ca7.5)before significant rehardening was found; there were no effects observedin the 4 hour groups. After treatment for 16 hours, SMH numberssignificantly increased in three out of the four specimens (P=0.001,P<0.003, P=0.015, respectively) (Table 1). The relative SMH after acidetching (Stage 2) was decreased to 80.6% of its original value and aftertreatment with the caseinate solution for 16 hours this increased to86.5% (Stage 3). The mean R was 32.1%.

The Effect of Phosphoprotein obtained by Partial Cross linking ofPartially Hydrolyzed Casein (PC) on Etched Enamel Surface

Table 1 shows the change of SMH numbers of enamel surface aftertreatment with the phosphoprotein preparation obtained by partial crosslinking of partially hydrolyzed casein (PC) with 36 mmol/L (PO₄)³⁻; 60mmol/L Ca²⁺ (PC-60Ca). The SMH numbers of etched-enamel weresignificantly increased after the 4 hours incubation in PC-60Ca with allten specimens. Seven out of ten specimens almost returned to the initial(before-etching) SMH values. The SMH numbers were significantly higherthan that of initial level in two tests (20%) (P=0.012) after treatmentwith PC-60Ca.

The relative SMH after acid etching (Stage 2) was decreased to 78.3% ofits original value and after treatment with the PC solution for fourhours this increased to 98.2% (Stage 3). The remineralisation yield (R)was 92.1% (n=10).

The exposure of acid etched enamel to the phosphoprotein solutionwithout Ca²⁺ (PC-noCa) or to calcium phosphate buffer with nophosphoprotein preparation (no PC-60Ca) resulted in no significantregain in hardness (Table 1).

Summary of the Performance of the Different Remineralisation Solutionson the Recovery Yield of the Etched Enamel Surface

The enamel remineralisation yield is presented in FIG. 1. The differencein remineralisation yield between solutions was clear. PC-60Ca produceddistinctly greater remineralisation potential than that of others.

De- and Remineralisation Assessment by Scanning Electron Microscopy(SEM)

Examination of the enamel surface by Scanning Electron Microscopy priorto etching showed the surface to be smooth (FIG. 2). In contrast, it wasfound that acid etching of sound enamel resulted in (1) a loss ofsurface enamel, (2) an increase in the tooth surface area due to theroughening of the tooth surface and (3) exposure of a more reactivesurface following the removal of superficial inert enamel (FIG. 3).

After treatment with PC-60Ca, the etched enamel surface was covered witha relative smooth and dense coating with frequent rod-shaped products.In most areas, a moderately uniform surface coating was present withadherent reaction products of 0.5-1 μm in length The coating wassufficiently dense to obscure the enamel prisms. The distribution of thedeposits was relatively homogeneous and the deposits covered almost allthe enamel surface (FIG. 4). The presence of small rod-shaped productswas assumed to be phosphoprotein-calcium phosphate complexes (PCCPC),though morphologic appearance alone does not identify a chemicalcompound. It can be seen that many rod-shaped products were present inthe demineralised interprismatic regions.

Discussion

Exposure of demineralised enamel surface to remineralisation solutionsshowed a regain in microhardness. This may indicate a partialrestoration of the calcium phosphate content. The net result was afilling up of intra- and interprismatic spaces, which was assesseddirectly by SEM morphological observation and indirectly by indentationlength measurements. A reduced porosity of the enamel surface in the SEMcauses an increased resistance to the indenter penetration into the testsurface, which is reflected by a smaller indentation length and suggeststhat remineralisation has occurred.

Remineralisation Effect of Saliva on Etched Enamel Surface

That saliva resulted in the rehardening of the etched enamel wasexpected as the remineralising ability of saliva is well reported(Koulourides et al. 1965, Leach et al. 1989, Peretz et al., 1990).Saliva can be described as “the bloodstream of the teeth”—being rich inminerals and proteins and supersaturated with respect to calcium andphosphate ions. It surrounds and bathes the tooth and provides aconstant supply of ions to the enamel surface (Peretz et al., 1990).When cleaned enamel is wet by saliva, specific proteins (such asstatherins and proline-rich proteins) from the saliva are adsorbed ontothe teeth surface to form the salivary pellicle or acquired pellicle.These two protein groups are thought to inhibit primary (spontaneous)and/or secondary precipitation (crystal growth) of calcium and phosphatefrom saliva. This appears to be a necessary and important activity inthe oral cavity because human saliva is supersaturated with respect tomost calcium phosphate salts. These precipitation inhibitors keep thesaliva in a state of supersaturation. The pellicle plays an importantrole in protecting the enamel by serving as a diffusion barrier.

An etching or carious lesion occurs in particular locations on theenamel surface where the equilibrium is upset and where a net loss ofmineral has occurred. The physical nature of the surface enamel, thesaliva and the acquired pellicle can be considered as analogous todefence mechanisms present in other systems in the body.

Remineralisation Effect of Phosphoproteins obtained by Partial Crosslinking of Partially Hydrolysed Casein (PC) and Caseinate on EtchedEnamel Surface

Under the experimental conditions, PC and caseinate were shown to coaton, incorporate in and reharden the etched enamel surface when assessedby microhardness testing and SEM. The SMH numbers of etched enamelshowed almost complete recovery after treatment with the PC solution.The SEM showed a dense coating layer was precipitated on the enamelsurface that resisted removal, even after 10 minutes of water washing.However, the crystalline nature of the surface enamel cannot bedetermined by the SEM method used in the present study.

The caseinate was not as effective as the PC and required a treatmenttime of 16 hours to achieve significant rehardening. The major reasonfor this was believed to be the low solubility of the caseinate in thepresence of calcium and phosphate ions.

For successful rehardening of the enamel when using the PC calcium andphosphate ions had to be present. The omission of any one of the threecomponents (phosphoprotein, calcium and phosphate) resulted in noremineralisation phenomenon being observed. Although the time period andchemical conditions (the conditions will be more complex in oralenvironment) are empirical, the key conclusion is that the de- andremineralisation occurred in this model system.

The possible reasons for the high remineralising ability of thephosphoprotein in conjunction with the calcium and phosphate ions are asfollows:

-   1) The hydrolysis process followed by the partial cross linking    process results in a protein with greater stability than that of    caseinate. The hydrolysis treatment followed by the partial cross    linking treatment appears to interfere with the self-associating    nature of casein and appears to inhibit its micelle-forming    tendency. Due to this decreased tendency to self associate the    protein will remain soluble in the presence of high levels of    calcium and phosphate.-   2) The calcium phosphate rich phosphoprotein is the main component    of the PC solution. The promotion of enamel remineralisation by the    PC is consistent with the protein solubilising calcium and phosphate    ions being at least partially responsible for remineralisation    activity of casein. It is thought that the PC increased the    solubility of calcium ions in solution, resulting in a higher    concentration of free calcium ions available into the pores of    etched enamel surface to facilitate remineralisation activity.-   3) Without wishing to be bound by any theory, the remineralisation    model of the phosphoprotein obtained by partial cross linking of    partially hydrolysed casein may be related to its ability to coat on    and incorporate in the etched enamel surface. The PC contains    closely situated groups of phosphoseryl residues that bind calcium    phosphate and hydroxyapatite very strongly. These sections of the    protein could calcium-bridge, ionically interact, and hydrogen-bind    with the enamel surface. On binding to the hydroxyapatite surface,    the protein coating may act as a reservoir, releasing calcium ions    available for use in remineralisation or redeposition into areas of    demineralisation in the crystal lattice. As shown in the present    study, casein phosphoprotein-calcium phosphate particles are present    on the enamel surface of the calcium-releasing bonding systems,    forming a potential protective deposit on the enamel surface.    Dissolution of calcium ions from casein phosphoprotein-calcium    phosphate complexes (CPPCPC) and diffusion into the pores in the    enamel may have occurred.

Example 4

Preparation of a Phosphoprotein (MAP 112)

Tryptic Hydrolysis

Fifty-five kilograms of sodium caseinate was dispersed in 50° C.deionised water so that a final concentration of 522L was achieved. Thesolution was cooled to 37° C., the pH adjusted to 7.06 with NaOH andporcine derived trypsin (Novo.4500K, molecular weight 23,400 Da,activity 4500 USP units/mg) added at 0.01% w/w casein and incubated forfive minutes. The solution was heated to 80° C. over 15 minutes, heldfor four minutes and cooled to 45° C.

Molecular weight profile after hydrolysis Molecular weight rangePercentage ≧30,000 10.7 <30,000, ≧21,000 57.8 <21,000, ≧12,000 15.7<12,000 15.8Transglutaminase Treatment

The pH was re-adjusted to 7.0, and transglutaminase (1% commercialpreparation, Activa MP, Ajinomoto Co) added at a ratio of 4.5% w/wcasein and incubated for 15 hours (temperature at the end of incubationwas 32° C.). The solution was heated to 80° C. and held for 5 minutes.The solution was diluted to 1000L and cooled to 5° C. The solution wasultrafiltered until a final concentration of 20% solids was achieved andthen spray dried. The molecular weight material greater than 30,000 Dawas increased by 100% and the number of cross-links in the protein was92 μmmol/g.

Example 5

Remineralisation of Enamel Following a Gentle Erosion Model

A second acid erosion model that provided less aggressive erosion wasexamined using an acetic acid-based system.

Methods

The enamel was prepared as per Example 3.

Preparation of Demineralisation Solution

24 and 48 Hours Demineralisation Solutions

Two demineralisation solutions were prepared at pH 4.5 and pH 4.8 with0.1 mol/L acetic acid and the pH adjusted with NaOH. Hydroxyapatite (HA)750 mg/L and 500 mg/L of were added to the demineralisation solutions atpH 4.5 and pH 4.8, respectively. Sodium azide (0.05 g/L) was added toeach solution as a preservative and the bottle wrapped with foil andstored at 4° C. until required.

72 hours Demineralisation Solutions

Sample preparation procedure as above sample, except that 750 mg/L and1.0 g/L of HA were added to the demineralisation solutions at pH 4.5 andpH 4.8, respectively.

Preparation of Remineralisation Solution

Twenty-five grams of MAP 112 protein (prepared as described in Example4) was dispersed in 300 mL of distilled water. A 2M calcium chloridesolution was added dropwise and then after 30 minutes the sodiumphosphate (Na₂HPO₄) was added so that a final concentration of 60 mmolCa²⁺ and 36 mmol PO₄ ⁹⁻ were achieved. The pH was adjusted to 7.0 using4M NaOH and made up to volume (500 ml). Sodium azide (0.05 g/L) wasadded to each solution as a preservative and the bottle wrapped withfoil and stored at 4° C. until required.

Protocol for Enamel Demineralisation

The specimens were demineralised in 25 ml of demineralisation solution(under constant shaking, in dark) for periods 24 hours, 48 hours and 72hours at 25° C.

Protocol for Enamel Remineralisation

The demineralised enamel blocks were remineralised in MAP 112 proteinsolutions containing 60 mmol/L Ca ions and 36 mmol/L phosphate ions. Theenamel blocks were immersed in 25 ml of remineralisation solution (underconstant shaking) for various time periods at 25° C. in the dark. Theremineralisation solution was changed every three days.

Assessment of Changes in the Enamel

Microhardness

The microhardness was measured as per Example 3 on the untreated enamel,enamel after demineralisation and at periods during remineralisation.

Results

On demineralisation all samples showed a decrease in surfacemicrohardness (SMH) with the pH 4.5 solution showing a bigger decreasethan the pH 4.8 solution. Surface changes were confirmed by lightmicroscopy. When the remineralisation portions of the graphs areexamined it can be seen that all samples show a slow increase in SMH. Asdiscussed in Example 3 mineral content of enamel shows a strong positiverelationship with SMH.

Example 6

Remineralisation of Enamel Treated to Induce Caries-Like Defects

Methods

The enamel was prepared as per Example 3.

Preparation of Demineralisation Solution

The demineralising solution was prepared following the method of WhiteDJ: Use of synthetic polymer gels for artificial carious lesionpreparation. Caries Res 21(3):228-242, 1987.

A polyacrylic acid/lactate solution, containing 0.2% polyacrylic acid(Carbopol C907) and 0.1 mol/L lactic acid was prepared as follows.Polyacrylic acid (1 g) was dissolved in 350 ml of distilled water atroom temperature, 4.29 ml of lactic acid solution (87.5%) was addedafter polyacrylic acid had dispersed, the pH was adjusted to 4.80 (4MNaOH) and made up the volume (500 ml) and transferred the solution to500-ml Schott bottle. Sodium azide (0.05 g/L) was added to each solutionas a preservative and the bottle wrapped with foil and stored at 4° C.until required.

A saturated hydroxyapatite, polyacrylic acid/lactate solution,containing 0.2% polyacrylic acid (carbopol C907) and 0.1 mol/L lacticacid was prepared as follows. Polyacrylic acid (1 g) was dissolved in350 ml of distilled water at room temperature, 4.29 ml of lactic acidsolution (87.5%) was added after polyacrylic acid had dispersed,followed by 3.7 g of hydroxyapatite, the pH was adjusted to 4.80 (4MNaOH) and made up the volume (500 ml) and transferred the solution to500-ml Schott bottle. Sodium azide (0.05 g/L) was added to each solutionas a preservative and the bottle wrapped with foil and stored at 4° C.until required.

The final demineralisation solutions were prepared immediately prior todemineralisation by diluting the saturated hydroxyapatiteCarbopol/lactate solution with the Carbopol/lactate solution with nohydroxyapatite.

About 55 ml of saturated hydroxyapatite Carbopol/lactate solution wascentrifuged at 2500 rpm for 20 minutes, the 50 mL of supernatant wascollected and mixed with 50 mL of the Carbopol/lactate solution with nohydroxyapatite. The pH was adjusted to 4.8.

Preparation of Remineralisation Solution at Three Different CalciumLevels

Three lots of 25 g of MAP 112 protein were dispersed in 300 mL ofdistilled water. A 2M calcium chloride solution was added dropwise andthen after 30 minutes the sodium phosphate (Na₂HPO₄) was added accordingto following table: Ca²⁺ (mM) 30 45 75 Weight (g) (as CaCl₂) 7.50 11.2518.75 PO₄ ³⁻ (mM) 18 27 45 Weight (ml) (as Na₂HPO₄) 1.278 1.916 3.194

Hydroxyapatite (500 mg/L) was added to each solution, the pH wasadjusted to 7.0 using 4M NaOH and made up to volume (500 ml). Sodiumazide (0.05 g/L) was added to each solution as a preservative and thebottle wrapped with foil and stored at 4° C. until required.

Protocol for Enamel Demineralisation

The specimens were demineralised in 25 ml of demineralisation solution(under constant shaking, in dark) at 25° C. for 96 hours.

Protocol for Enamel Remineralisation

The enamel blocks were remineralised in MAP 112 protein solutionscontaining 30, 45, 60 or 75 mmol Ca ions. The enamel blocks wereimmersed in 25 ml of remineralisation solution (under constant shaking)for various time periods at 25° C. in the dark.

Assessment of Changes in the Enamel

Microhardness

The microhardness was measured as per Example 3 on the untreated enamel,enamel after demineralisation and at periods during remineralisation.

Example 7

Streptococcus mutans Adhesion Assay

Method

Streptococcus mutans NCTC 10449 was grown in trypone/yeast extractmedium containing brain/heart infusion and radiolabelled with[³H]thymidine. Hydroxyapatite beads (20 mg, Biorad) were hydrated in KClbuffer (Cannon et al., 1995) and then incubated with pooled human saliva(30% v/v KCl buffer). The beads were washed with KCL buffer and then thesaliva-coated hydroxyapatite beads were incubated with radiolabelled S.mutans cells. Non-adherent S. mutans cells were removed from the beadsby washing with KCl buffer and then the percentage of the cells added toeach assay that adhered to the SHA beads were calculated.

MAP 111 Phosphoprotein (DH 5.0, 140 μmol cross-links/g protein) wasadded at the following concentrations: 0.001%, 0.01%, 0.1%, 1.0% and10.0% (in KCl buffer). The effect of the phosphoprotein on adhesion,relative to assays containing no phosphoprotein was calculated.

Each assay was conducted in triplicate and each experiment was conductedthree times.

It can be seen from FIG. 14 that the protein effectively inhibitedadhesion of S. mutans at concentrations as low 0.01% and still achievedabout 40% inhibition of adhesion at 0.001% protein. For the cariesprocess to be initiated S. mutans needs to adhere to the tooth surface.Hence by reducing the number of S. mutans adhering the risk of cariesformation can be reduced. These assay results illustrate that theproteins not only possess a reparative function but also a preventativefunction.

Reference: Cannon, R. D., Nand, A. K and Jenkinson, H. F. (1995)Adherence of Candida albicans to human salivary components adsorbed tohydroxyapatite. Microbiology 141:213-219

Example 8

Measurement of Iron Binding Capacity of the Phosphoprotein Preparations

The iron binding capacity of the proteins was determined byre-suspending the protein in water, adding FeCl₂ under constant pH;removing the insoluble material (salts and protein); then removing thesoluble non bound salts and determining the amount of calcium bound tothe soluble protein. The experimental details were as follows.

A 1% solution of the proteins were dissolved with milli-Q water, andallowed to stand for 1 hour to ensure complete hydration. Ferrouschloride was added at the following levels: 0 mM, 10 mM, 30 mM, and 40mM; and the solution incubated at 25° C. for 1 hour with good mixing.Throughout the experiment the pH was maintained at 7.0 using NaOHsolution. The samples were incubated at 25° C. for 6 to 10 hours withgood mixing. After incubation, a sample was centrifuged at 10 000×g for10 minutes and filtered through a 0.2 μm nylon filter.

The sample was injected into a 2 ml sample loop and loaded onto aPharmacia FPLC fitted with Sephadex G-25 (Vt-25 ml) desalting column.The running buffer was 10 mM HEPES at pH 7 the flow rate was 2 ml/minand detection was achieved through UV absorption (280 ηm), conductivityand pH. The protein peak was collected and iron concentration determinedby atomic absorption spectroscopy (AAS).

Iron Binding Capacity of Lot 6 after Cross-Linking with Tg for 18 hours.Expressed as mg Fe²⁺/g Initial Protein, and mg Fe²⁺/g of SolubleProtein. mg Fe²⁺/g protein mg Fe²⁺/g protein [Fe²⁺] (total) (soluble)  0mM 0.0 0.0 10 mM 2.9 6.4 30 mM 3.5 13.8 40 mM 3.7 19.5

Fe²⁺ was found to bind to the Lot 6, 18 hr Tg phosphoprotein preparationat a maximum of 3.7 mg Fe²⁺/g protein. Of the protein that remainedsoluble, the Fe²⁺ bound at about 20 mg Fe²⁺/g soluble protein.

Zinc, Iron II and Magnesium Binding Capacity of Phosphoproteins mmolions added Zinc ions Iron II Magnesium ions 5 22.2 21.7 4.6 10 41.0 44.85.4 15 43.0 44.0 5.4

Example 9

Phosphoprotein Fortified Processed Cheese

The following two processed cheeses were prepared: % weight/weightFormulation 1 2 Processed cheese 95.60 95.42 MAP (phosphoprotein) 1.341.34 Natural milk calcium phosphate 2.87 2.86 Calcium chloride 0.00 0.19Water 0.19 0.19

The cheeses were prepared in a Brabender W50 mixer at 45° C. and 50 RPM.The processed cheese (1 cm³ cubes) was fed into the mixer over about 60seconds, the water added and mixed for three minutes. The preblendednatural milk calcium phosphate and MAP 112 were added and mixed for tenminutes. The mixer was stopped, the cheese mixture was removed, formedinto a block, wrapped in cling film and stored at 5° C. until required.

Remineralisation Potential of Modified Processed Cheese.

The enamel was prepared as per Example 3, the demineralisation wasconducted as per Example 6 and the remineralisation was as per Example 6except that a cheese slurry preparation was used in place of theremineralising solution. The microhardness was measured as per Example3.

Cheese Slurry Preparation

The modified processed cheeses contained about 20% protein almost all ofwhich was casein. Fifty-five grams of modified processed cheese wasweighed and blended (with Waring Blender, high speed) with 144 ml ofdistilled water for 5 minutes at room temperature. The cheese slurry wascentrifuged for 20 minutes at 4,350 g and the aqueous solution (middlelayer) and the fatty supernatant were carefully transferred to aseparating flask. The mixture was left to separate and the aqueous phaseremoved. Sodium azide (0.05 g/L) was added to each solution as apreservative and the bottle wrapped with foil and stored at 4° C. untilrequired.

Results

Microhardness

The demineralisation achieved a good reduction in surface hardness andas mentioned earlier this is strongly correlated with enamel mineralconcentrations. On remineralisation a slow increase in SMH can be seen.This increase in SMH suggests the enamel is being remineralised.

Example 10

Remineralising Emulsion Formulation Ingredients % weight/weightPhosphoprotein MAP112 10.00 Natural Milk Calcium Phosphate (Alamin) 7.50Sorbitol 4.50 GMS 400V 1.00 Propyl paraben 0.075 Methyl paraben 0.025Medium chain triglycerides 7.50 Glycerol 1.00 Delios S 30.00 Xanthan gumsolution (0.1%) 38.30 Peppermint Oil 0.074Method

The dry ingredients (MAP, Alamin, Sorbitol, Methyl and Propyl Paraben)were blended and dispersed the maltitol syrup and Xanthan gum solution.The GMS and Glycerol were heated to about 80° C. (until molten), themedium chain triglycerides were added and heating maintained until themixture was lump-free. The aqueous phase was heated to about 70° C., theoil phase was added to aqueous phase with continuous stirring. ThePeppermint Oil was added, the mixture heated to 90° C. (held for 5minutes) and then homogenised (Ultraturrex). The mixture was hot filledinto a clean sterile bottle, rinsed with 96% ethanol and stored at 5° C.until required by patients.

The patients found that the emulsion provided a good coating on theinside of their mouth and provided good wetting.

Example 11

Candida albicans Adhesion Assay

A. Method

Candida albicans ATCC 10261 was grown in GSB medium (glucose, salts,biotin, Cannon et al, 1995) and radiolabelled with [³⁵S]methionine.Hydroxyapatite beads (20 mg, Biorad) were hydrated in KCl buffer (Cannonet al., 1995) and then incubated with pooled human saliva (30% v/v KClbuffer). The beads were washed with KCL buffer and then thesaliva-coated hydroxyapatite beads were incubated with radiolabelled C.albicans cells. Non-adherent C. albicans cells were removed from thebeads by washing with KCl buffer and then the percentage of the cellsadded to each assay that adhered to the SHA beads was calculated.

MAP 111 protein (DH 5.0, 140 μmol cross-links/g protein) was added atthe following concentrations: 0.1%, 1.0% and 10.0% (in KCl buffer). Theeffect of the MAP protein on adhesion, relative to assays containing noMAP protein was calculated.

Each assay was conducted in triplicate and each experiment was conductedthree times.

B. Results

It can be seen from FIG. 13 that the protein inhibited adhesion ofCandida albicans at concentrations as low 0.1%. The inhibition appearsto dose-dependent.

Reference:

Cannon, R. D., Nand, A. K. and Jenkinson, H. F. (1995) Adherence ofCandida albicans to human salivary components adsorbed to hydroxapatite.Microbiology 141:213-219

Example 12

Determination of Degree of Cross Linking Using High Performance LiquidChromatography

The following method was used to determine the degree of cross linkingof the phosphoprotein preparations, in terms of the number ofglutamyl/lysl bonds.

Chemicals and Reagents

ε-(γ-Glutamyl)lysine (G-L) and trifluoroacetic acid (TFA, proteinsequencing grade), Prolidase (porcine kidney), Leucine aminopeptidaseand cytosol from hog kidney, Carboxypeptidase A (bovine pancreas) andPronase (Streptomyces griseus) and TRIS[Tris(hydroxymethyl)aminomethane] were purchased from Sigma Chem. Co.(Sydney, Australia). Acetonitrile (HPLC grade) was purchased from Biolab(Christchurch, New Zealand).

Proteolytic Digestion of the Cross-Linked Proteins

A 48-50 mg aliquot of protein sample was weighed in a glass test tube(total volume 15 mL). A crystal of thymol and 2 ml of 0.2 M Tris (pH8.0, HCl) was added, the solution vortexed and then incubated at 40° C.for 1 h to allow the dispersion of the protein. An aliquot of Pronase(0.4 U/mg protein) was added to the mixture which was then incubated at37° C. for 24 h. The pronase digestion was continued for a further 24 hby the addition of another equal sized aliquot of pronase. Afterinactivation of pronase by heating at 100° C. (waterbath) for 10 min,the digestion was continued adding leucine aminopeptidase (0.4 U/mgprotein), the solution being treated as for the pronase incubation. Thedigestion was continued using prolidase (0.45 U/mg protein) and thencarboxypeptidase A (0.2 U/mg protein). After final inactivation themixture was diluted to 7.5 g with ultrapure water (MilliQ waterpurification system)(Millipore, North Ryde, Australia).

HPLC Analysis of G-L

The HPLC system consisted of a Dionex GP40 gradient pump solventdelivery system connected to an ICI Instruments 1210 UV/Vis detector.Data was captured via an ITNS Acquisition Board and analysed using theAZUR chromatography software Version 1.1. The samples (100 μl) wereseparated on an Inertsil ODS-2 column (5 μm, 150×7.6 mm) (Phenomenex,Aucldand, New Zealand) connected to a guard column (C₁₈ ODS, 4 mm×3.0 mmID)(Phenomenex) and a 2 μm prefilter. Analysis was performed at 2.5° C.The mobile phases were 0.1% TFA (v/v)(solvent A) and acetonitrilecontaining 0.1% TFA (v/v) (solvent B). The solvent program was asfollows: 100% solvent A for 20 min, 0% to 100% solvent B from 20 to 25min, 100% solvent B from 25 to 50 min. The detector wavelength was setat 210 nm and the flow rate was 1.0 ml/min.

All samples from proteolytic digestion were filtered on a 0.45 μmMillex-HA Millipore filter unit. 200 μl of sample was mixed with 100 μlof distilled water and 100 μl TFA (2% w/w). The G-L peak was identifiedby comparison to elution time of a standard and confirmed by standardaddition of a G-L standard solution to the sample.

Example 13

Method of Determining Degree of Hydrolysis of Partially HydrolysedCasein

Samples were prepared as either 0.1% or 1% protein (w/v) solutions indistilled water. A 100 μl aliquot of sample was added to 800 μl of0.2125 M sodium phosphate buffer, pH 8.20. To this 800 μl 0.1% TNBSreagent was added, and the reagents were mixed well, wrapped in foil andincubated at 50° C. in a covered water-bath. After exactly 60 minutesthe reaction was terminated by the addition of 1600 μl of 100 mM HCl andthe samples left to cool to room temperature for 30 minutes beforereading absorbance against a buffer/TNBS blank at 340 ηm.

INDUSTRIAL APPLICATION

It is believed that the methods and compositions of the presentinvention will find application in delivering bioactive metal ions, suchas calcium and iron, to mammals. The invention is also expected to findparticular application in compositions for remineralising teeth and/orfor preventing or treating dental caries, tooth erosion, dentinalsensitivity or gingivitis.

The present invention is believed to possess certain advantages overteeth remineralising/anticaries compositions comprising phosphopeptidesobtained from partial hydrolysis of casein, such as those described inU.S. Pat. No. 5,015,628. In particular, the phosphoprotein preparationsused in the present invention have a clean flavour, in contrast topartial casein hydrolysates, which contain bitter-tasting hydrophobicpeptides that need to be removed in order for the product to have anacceptable flavour. In addition, the phosphoprotein preparations of thepresent invention utilize the vast majority of the proteinaceousmaterial from the casein (rather than just the proportion containing thecasein phosphopeptides), thereby reducing wastage.

The phosphoprotein preparations used in the present invention also haveadvantages over remineralising/anticaries compositions containingunmodified casein or caseinate; in particular, the calcium-binding andteeth remineralising properties of the phosphoprotein preparations are,at least in preferred embodiments, believed to be significantly greaterthan those of casein. In addition, the phosphoprotein preparations arerelatively soluble and have a lower viscosity than unmodified casein,thereby facilitating their incorporation into compositions.

Although the present invention has been described with reference toparticular embodiments, those persons skilled in the art will appreciatethat numerous variations and modifications may be made without departingfrom the scope of the invention as defined in the following claims.

1. A composition for delivering a bioactive metal ion to a mammal, the composition comprising an effective amount of a source of the bioactive metal ion, a phosphoprotein preparation obtained by partially cross linking a partial hydrolysate of casein or a caseinate, and one or more physiologically acceptable diluents or carriers.
 2. A composition according to claim 1, wherein the metal ion is divalent.
 3. A composition according to claim 1, wherein the metal ion is selected from the group consisting of calcium, iron, zinc, cobalt, copper and magnesium.
 4. A composition according to claim 1, wherein the composition is an oral composition and comprises a foodstuff, beverage, or a pharmaceutical vehicle.
 5. A composition according to claim 1, wherein the pH of the composition is between about 6 and about
 9. 6. A composition according to claim 1, wherein the composition comprises a processed cheese product, and the source of a bioactive metal ion comprises natural milk calcium phosphate.
 7. A composition according to claim 1, wherein the partial hydrolysate has been obtained by enzymatic hydrolysis of acid casein, rennet casein or a caseinate.
 8. A composition according to claim 7, wherein the enzyme is trypsin.
 9. A composition according to claim 7, wherein the degree of hydrolysis is in the range of about 3% to about 8% of the total number of peptide bonds.
 10. A composition according to claim 9, wherein the degree of hydrolysis is in the range of about 3.5% to about 7%.
 11. A composition according to claim 9, wherein the degree of hydrolysis is in the range of about 4% to about 6.5%.
 12. A composition according to claim 7, wherein the degree of hydrolysis is such that about 10% or less of the casein or caseinate is rendered insoluble at pH 7, by the partial hydrolysis.
 13. A composition according to claim 12, wherein the degree of hydrolysis is such that about 5% or less of the casein or case mate is rendered insoluble at pH 7, by the partial hydrolysis.
 14. A composition according to claim 1, wherein the partial hydrolysate has been partially cross linked enzymatically, using the enzyme transglutaminase.
 15. A composition according to claim 1, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises about 10 or more μmol cross links per gram of protein.
 16. A composition according to claim 1, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises between about 10 and about 250 μmol cross links per gram of protein.
 17. A composition according to claim 1, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises between about 50 and about 160 μmol cross links per gram of protein.
 18. A method of delivering metal ion to a mammal, comprising administering to the mammal a composition according to claim
 1. 19. A composition for remineralizing tooth enamel and/or for treating or preventing one or more conditions selected from the group consisting of dental caries, tooth erosion, dentinal hypersensitivity and gingivitis in a mammal, wherein the composition comprises an effective amount of a phosphoprotein preparation in combination with one or more carriers or diluents, wherein the phosphoprotein preparation has been obtained by partially cross linking a partial hydrolysate of casein or a caseinate.
 20. A composition according to claim 19, wherein the partial hydrolysate has been obtained by enzymatic hydrolysis of acid casein, rennet casein or a caseinate.
 21. A composition according to claim 20, wherein the enzyme is trypsin.
 22. A composition according to claim 19 wherein the degree of hydrolysis is in the range of about 3% to about 8% of the total number of peptide bonds.
 23. A composition according to claim 22, wherein the degree of hydrolysis is in the range of about 3.5% to about 7%.
 24. A composition according to claim 22, wherein the degree of hydrolysis is in the range of about 4% to about 6.5%.
 25. A composition according to claim 19, wherein the degree of hydrolysis is such that about 10% or less of the casein or caseinate is rendered insoluble at pH 7, by the partial hydrolysis.
 26. A composition according to claim 25, wherein the degree of hydrolysis is such that about 5% or less of the casein or case mate is rendered insoluble at pH 7, by the partial hydrolysis.
 27. A composition according to claim 19, wherein the partial hydrolysate has been partially cross linked enzymatically, using the enzyme transglutaminase.
 28. A composition according to claim 19, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises about 10 or more μmol cross links per gram of protein.
 29. A composition according to claim 28, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises between about 10 and about 250 μmol cross links per gram of protein.
 30. A composition according to claim 28, wherein the degree of partial cross linking is such that the resulting phosphoprotein preparation comprises between about 50 and about 160 μmol cross links per gram of protein.
 31. A composition according to claim 19, further comprising a source of calcium ions.
 32. A composition according to claim 31, wherein the composition further comprises a source of phosphate ions.
 33. A composition according to claim 30, wherein the composition further comprises calcium phosphate.
 34. A composition according to claim 30, wherein the composition further comprises natural milk calcium.
 35. A composition according to claim 31, wherein calcium ions are present in the composition at a level of at least about 5 mmol calcium ions per gram of phosphoprotein preparation.
 36. A composition according to claim 35, wherein calcium ions are present in the composition at a level of at least about 10 mmol calcium ions per gram of phosphoprotein.
 37. A composition according to claim 32, wherein the molar ratio of calcium ions to phosphate ions is in the range of about 0.8-1.2:0.4-0.8.
 38. A composition according to claim 37, wherein the molar ratio of calcium ions to phosphate ions is about 1:0.6.
 39. A composition according to claim 19, wherein the composition further comprises a source of strontium ions.
 40. A composition according to claim 39, wherein the composition further comprises a source of fluoride ions.
 41. A composition according to claim 19, wherein the composition comprises a composition selected from the group consisting of a mouthwash, a dentifrice, toothpaste, a powder, an emulsion and a gel.
 42. A composition according to claim 19, wherein the composition comprises an emulsion, wherein the phosphoprotein preparation is present in an amount of about 1% to about 15% by weight of the emulsion, and the emulsion further comprises natural milk calcium phosphate, in an amount of about 3% to about 12% by weight of the emulsion.
 43. A composition according to claim 19, wherein the composition comprises a foodstuff and a confection.
 44. A method for remineralising tooth enamel and/or for treating or preventing one or more conditions selected from the group consisting of dental caries, tooth erosion, dentinal hypersensitivity and gingivitis in a mammal, the method comprising contacting the teeth of the mammal with a composition according to claim
 19. 45. A phosphoprotein preparation, which has been obtained by partially cross linking a partial hydrolysate of casein or a caseinate, and wherein the degree of partial hydrolysis of the casein or case mate prior to cross linking is in the range of about 3% to about 8% of the total number of peptide bonds, and the degree of partial cross linking is such that the phosphoprotein preparation comprises about 10 or more μmol cross links per gram of protein.
 46. A phosphoprotein preparation according to claim 45, wherein the degree of partial hydrolysis of the casein or caseinate prior to cross linking is in the range of about 3.5% to about 7%.
 47. A phosphoprotein preparation according to claim 45, wherein the degree of partial hydrolysis of the casein or caseinate prior to cross linking is in the range of about 4% to about 6.5%.
 48. A phosphoprotein preparation according to claim 45, wherein the degree of partial cross linking is such that the phosphoprotein preparation comprises between about 10 and about 250 μmol cross links per gram of protein.
 49. A phosphoprotein preparation according to claim 45, wherein the degree of partial cross linking is such that the phosphoprotein preparation comprises between about 50 and about 160 μmol cross links per gram of protein.
 50. A phosphoprotein preparation according to claim 45, wherein the degree of hydrolysis is such that about 10% or less of the casein or caseinate is rendered insoluble at pH 7, y the partial hydrolysis.
 51. A phosphoprotein preparation according to claim 45 wherein the degree of hydrolysis is such that about 5% or less of the casein or caseinate is rendered insoluble at pH 7, by the partial hydrolysis. 